Conductance detection systems and methods may be used in a wide variety of applications—one important example being separation methods and apparatus. Separation methods and apparatus are important in many contexts, including in the fundamental sciences such as chemistry, biology, environmental sciences, and the like as well as in industries relating to pharmaceuticals, health, chemicals, petroleum, food and the like. By using a separation method and apparatus, a sample mixture comprising a plurality of species is converted into two or more distinct products.
Many variations of separation methods and related apparatus are well known in the arts. By way of example, they may involve transporting a sample mixture using a mobile phase, which may be a liquid or a gas as in chromatography. Different species may be separated based on their interaction with a stationary phase, which may be disposed in a substantially columnar, capillary or planar geometry. By way of another example, if species in a sample mixture are charged, they may also be transported by an electric field, and different species may be separated based on their respective mobilities, as in electrophoresis. Varieties of electrophoretic separation methods and apparatus include those based on gels, capillaries, micro channels, 2-dimensional separations, etc. By way of yet another example, a species in a sample mixture in a liquid phase may be separated by being immobilized on a substrate, which then is removed from the sample mixture. This approach is central to many biosensors.
As various species are separated, there is a need to detect them for a variety of reasons, e.g. to evaluate the degree of separation (or lack thereof), to analyze them with respect to their quantity and their other properties, to aid in their isolation potentially for further processing or use, etc. Various types of detectors have been employed in this regard. With respect to separation methods and apparatus in which sample mixtures of species are carried by a liquid mobile phase, the most widely used detectors are based on optics. For species that have a significant absorbance in the ultra-violet to visible (UV-Vis) range of the electromagnetic spectrum, UV-Vis absorption spectroscopy may be employed.
It is noteworthy, however, that many important species such as carbohydrates, alcohols, and certain important polymers (for example, polyethylene glycol) do not have significant UV-Vis absorption. Further, application of UV-Vis spectroscopy to capillary electrophoresis, in particular, is challenging since UV-Vis absorbance is proportional to a path length over which light and a sample mixture interact but there is a desire to make capillary diameters small in order to yield improved separations.
The next most widely used type of detector is based on refractive index, that is, the ratio of the speed of light in a material relative to that in vacuum. Since index of refraction is a universal property of matter, even species that have no UV absorption are detectable. However, when detecting species that are good UV absorbers, UV-Vis detectors have much better sensitivity than index of refraction detectors. Also, index of refraction detectors are sensitive to temperature and require suitably controlled temperature environments.
Given a choice of various liquid mobile phases, if the composition of the liquid mobile phase is varied to elute a species from a column (as in gradient elution in high performance liquid chromatography or HPLC), the index of refraction of the solvent changes significantly, making additional potentially small changes in index of refraction due to species eluting from the column difficult to detect. That is, the large change in background due to changing solvent composition requires that the detector have a large dynamic range (achieved by coarse resolution). As a result, after this large change in background is subtracted from the data, smaller changes due to species have poor signal-to-noise. Incompatibility of refractive index detectors with gradient elution is a significant limitation hampering more wide spread application of refractive index detectors. In another optics-based approach, species may be excited (e.g. by a laser), and fluoresced light emitted by the species may be detected.
In cases where a species neither has significant UV-Vis absorbance nor generates significant fluorescence, the species may be chemically modified to increase its UV-Vis absorbance or fluorescence. This requires additional chemical processing steps, which can be undesirable due to a need for additional input of resources. Such additional chemical processing are common place, for example, when using thin layer chromatography or gel electrophoresis, and they may require significant additional processing time that is on the order of the time required to perform a separation itself. Furthermore, generally, detection methods and apparatus based on optics tend to be costly and somewhat unwieldy, hindering portability.
Therefore, for these reasons and others, other detection apparatus and methods not based on UV-Vis absorbance, refractive index or fluorescence have been developed. With respect to separation methods and apparatus that employ a liquid mobile phase, these include detection apparatus and methods based on electrochemical current, evaporative light scattering and mass spectroscopy. If a species can be dissolved in an electrolyte solution and can be oxidized or reduced, an electrochemical detector can be used to detect the species via the current produced by oxidation or reduction.
If a species is much less volatile than a solvent in which it is dissolved, then evaporative light scattering detectors can be used. In this approach for detecting a species, the species is atomized and transported by a gas. As the solvent evaporates, species form fine particles in the gas and are detected by light scattering. In liquid chromatography-mass spectroscopy, usually, the solvent is much more volatile than the species. Often volatile acids, bases or buffers are included in the sample mixture, and the species exists as ions in the sample mixture. Electrospray ionization is used to generate charged droplets of a sample mixture. As the droplets evaporate, eventually charged species remain and are detected by a mass spectrometer.
While these various methods have found useful applications, they also have significant limitations. Use of conductivity detectors is restricted to solvents that possess a practically measurable conductivity. Important organic solvents widely used with HPLC, e.g. hexane, have conductivities that are too low to be practically measurable. Also, if electrodes that probe conductivity are in contact with the sample mixture, changes in conductivity at the electrode-sample mixture interface can undesirably influence the overall conductivity measured. Electrochemical detectors are restricted to species that can be oxidized or reduced and to electrolytic solutions. Evaporative light scattering requires use of solvents that are much more volatile than species in the sample mixture. Important volatile molecules, for example, low molecular weight polymers such as polyethylene glycol, can not be detected. Similar restrictions arise in the case of mass spectroscopy detectors.
Mass spectroscopy detectors also require successful ionization of species and are typically very costly. Further, a number of the above-described detectors comprise myriad components such as optics, vacuum components, magnets, gas supplies, diode array detectors and the like that are bulky, require careful alignment, and thus significantly limit portability of the detectors. They also tend to be costly. Therefore, an apparatus and method for detecting species in a sample mixture that can detect a wide range of species, can function using a wide range of solvents (including solvents with varying composition that are employed during gradient elution), are easily portable and are cost effective are desired.
Although separation methods and apparatus using liquid mobile phase are desirable given that species are frequently synthesized in liquids, gas chromatography and associated detectors may also be employed to separate species. In this case, the most common apparatus and methods for detecting species are based on flame ionization detection and thermal conductivity detection. Both are sensitive to a wide range of components, and both work over a wide range of concentrations. Flame ionization detectors are sensitive primarily to hydrocarbons, and are more sensitive to them than thermal conductivity detectors. However, a flame ionization detector has difficulty detecting water. Other detectors are sensitive only to specific types of substances, work well only in narrower ranges of concentrations, may have limited portability and may be very costly. Other methods and apparatus for detecting species using gas chromatography include those based on discharge ionization, electron capture, flame photometry, Hall electrolytic conductivity, helium ionization, a presence of nitrogen phosphorus, photo-ionization, pulse discharge ionization, thermal energy analysis and mass spectroscopy.
Methods and apparatus for detecting species based on conductance measurements (or equivalently impedance measurements) provide an alternative to the above mentioned methods and apparatus, respectively. Conductance detectors make use of electronics which can be fabricated inexpensively and which can be compact and portable. Also, electrodes used to measure conductance probe the conductance of regions dictated by geometrical factors including electrode sizes, shapes, relative positions, and relative orientations, all of which are controllable over a wide range of length scales, from a nanometer length scales to micron length scales to millimeter lengths scales and even larger length scales. Electrodes can be fabricated exploiting chemical methods, electron beam lithography, optical lithography, shadow mask methods, and other methods well known to those skilled the arts. As such, methods and apparatus based on conductance detections are conveniently compatible with methods and apparatus for microfluidics, respectively, which enable extremely low sample and solvent volumes, sharp detection peaks, efficient separations and significant cost savings. Furthermore, conductance detection is amenable to methods well known in the arts for improving signal-to-noise, including lock-in detection and the like.
In a conductance measurement, a drive (such as a current or voltage) is applied to one or more electrodes and induces a response (such as a voltage or current, respectively). The drive may vary in time or may be substantially time independent. The sample mixture is arranged to traverse a proximity of at least one of the electrodes. The electrodes and sample mixture may or may not be in direct electrical contact with each other. For example, if the drive is a voltage, the one or more electrodes may cause a so-called external current, iext, to flow across surface(s) of the one or more electrodes by inducing mobile charges to flow across the surface(s); and/or, the one or more electrodes may cause a displacement current, idisp, to flow by charging or discharging as a function of time.
In a linear approximation, if a voltage difference, ΔV, is applied between a pair of electrodes and no displacement current flows between the pair of electrodes, the external current that flows between the electrodes is proportional to ΔV and is given byiext=ΔVGσ.  (1)If one neglects fringing effects, thenGσ=σ∫dA/L  (2)where Gσ is σ-conductance, σ is conductivity and depends on type and concentration of species present, dA is an element of cross section area through which the external current flows, and L is the distance over which the external current flows through the element. More generally,Gσ=σLσ  (3)where Lσ has dimensions of length and increases both with increasing cross sectional area and decreasing distance over which current flows. From Equation (3), it is evident that Lσ is a geometrical amplification factor for conductivity.
Also in a linear approximation, if a voltage difference, ΔV, is applied between a pair of electrodes and no external current flows between the pair of electrodes, the displacement current that flows between the electrodes is proportional to ΔV and is given byidisp=ΔVG∈.  (4)If one neglects fringing effects, thenG∈=jω∈∫dA/L  (5)where G∈ is ∈-conductance, j is a complex number such that j2=−1, ω is the angular frequency of the voltage difference assumed to vary sinusoidally with time, ∈ is a permittivity, dA is an element of cross sectional area over which the displacement current flows, and L is the distance over which the displacement current flows through the element. More generally,G∈=jω∈L∈  (6)where L∈ has dimensions of length and increases both with increasing cross sectional area and decreasing distance over which current flows. From Equation (6), it is evident that L∈ is a geometrical amplification factor for permittivity.
For sufficiently small voltages, the above-mentioned linear approximations work very well. A response of a conductance detector can frequently be adequately modeled by a net conductance, G, that is given as a series/parallel combination of suitable Gσ's and G∈'s by elementary circuit theory. Current can be measured by methods and apparatus well known in the arts and, given ΔV, G can be thereby determined.
G∈ and/or Gσ may vary depending on the type and concentration of species interacting with the one or more electrode. Detectors relying substantially on Gσ to detect species require that a presence of the species results in a practically detectable conductance change. With respect to chromatography, such Gσ detectors are commercially available. However, frequently (for example, in liquid chromatography) one employs solvents (e.g. hexane) which have conductivities that are too low to be practically measurable; as a result, species in such solvents can not be detected.
It is noteworthy that species that are amenable to electrophoretic separation are necessarily charged, and sample mixtures typically induce practically measurable conductance changes. Kuban (2004), Matysik (2008), Kuban (2008) and Pumera (2007) provide recent reviews of conductance detectors that detect a presence of a species in a sample mixture being separated via capillary and microchip electrophoresis via changes the species induce in Gσ. We refer to such detectors as Gσ detectors. Gσ detectors for capillary electrophoresis are generally classified according to whether or not electrodes are in direct electrical contact with the sample mixture. In the case that electrodes are in contact with the sample mixture, great care must be exercised to ensure that the electrodes to not adversely affect the forces that drive the species in the sample mixture. Gσ detectors for microchip electrophoresis may be similarly classified. They may also be classified according to whether electrodes are on-column (i.e. located on a separation channel in which species are separated), off column (i.e. located on a channel branching off a separation channel) or end-column (i.e. located at the end of the separation channel). As an exception to the latter classification, Wang (2003) disclosed electrodes that are movable along the separation channel and that, therefore, enable monitoring separation of species at various points along the separation channel. Clarke et al. in U.S. Pat. No. 5,194,133 disclose a microchip electrophoresis apparatus with an array of electrodes in contact with the sample mixture. However, the electrodes detect electrochemical current, rather than changes in Gσ. Tanyanyiwa et al. (2002) disclose that a Gσ detector with electrodes separated by ˜1 mm yields better signal when the separation channel is located ˜0.2 mm rather than ˜1 mm below the electrodes.
U.S. Pat. No. 4,301,401 by Roof and Benningfield teaches a dielectric constant detector having a sample cell and a reference cell to provide an electrical signal that is proportional to the concentration of a component being passed through the dielectric constant detector. The sample and reference cells are adjusted in such a manner that the capacitance of each cell is substantially equal when the same fluid is in both cells.
Electronic circuitry associated with each cell provides an output signal which has a frequency and which is a function of the capacitance of each cell, respectively. The two output signals are mixed to provide a difference frequency and the difference frequency is converted to a voltage to provide an electrical signal which is representative of the concentration of the particular species of the sample mixture which is passing through the sample cell. However, Benningfield et al. (1981) teach that the detector's oscillation quenches when an equivalent parallel resistivity of the solute/solvent becomes less than 0.27 MΩ-cm.
As a result, common solvents such as water can seldom be used due to impurities. The detector also can not be used with common buffers, salts or other electrolytic solutions. Further, the detector is incompatible with use of gradient elution. Also, the architecture of the conductance detector is not rigid. The mobile phase and sample mixture flow between capacitor plates such that changes in pressure and carrier flow rate cause undesirable and significant variations in L∈. Variations in L∈ caused by pressure make variations in ∈ caused by a species of interest more difficult to detect. Further, L∈ disclosed by prior art are small.
M. Yi et al. (2005) disclose a nanogap dielectric biosensor with an area of 1.5 μm×4 mm and an electrode separation of 20 nm; that is, the geometrical amplification factor is 1.5 μm×4 mm/20 nm or 30 cm. S. Roy et al. (2009) disclose mass-produced nanogap sensor arrays for ultrasensitive detection of DNA. The sensors are 5 μm×5 μm in area and the electrodes have a separation of 5 nm. In these sensors, only fringe electric fields are accessible for sensing. Assuming the fields extend beyond the edges of the sensor on a length scale that is on the order of a few times the electrode separation, the geometrical amplification factor is a few times 4 edges×5 μm×5 nm/5 nm or a few times 20 μm. The 819 Advanced IC detector (a conductivity detector) sold by Metrohm possesses cell constants, defined byCell constant=L/A,  (7)that range from 13 to 21 cm−1. The corresponding geometrical amplification factors range from 0.5 mm to 0.8 mm. Other conductivity detectors sold by Metrohm possess cell constants that range from 0.1 cm−1 to 10 cm−1 and corresponding geometrical amplification factors that range from 0.1 cm to 10 cm. Hollis et al in U.S. Pat. No. 5,846,708 disclose an optical and electrical apparatus for molecule detection. In FIG. 4 of this patent, they disclose a conductivity detector with electrodes that have footprints that do not overlap. Hence, sensing is performed by fringe fields, as shown in the figure. The geometrical amplification factor thus is a few times 50 lines×2 edges per line×100 μm×400 nm/400 nm or 1 cm. Such small geometrical amplification factors result in correspondingly small conductance changes caused by a presence of species and require correspondingly large amplification. Potentially small conductance changes are difficult to detect, may require careful post-signal processing and are generally vulnerable to noise.
Thus, there is generally a need for improved methods and detectors for measuring changes in conductance with much higher sensitivity than is currently available. Equations (3) and (6) indicate that increasing geometrical amplification factors can yield correspondingly improved methods and detectors for measuring changes in conductance. In principle, this can be accomplished by combining a large number of area elements increasing the cross-sectional area and by decreasing the length of the region interrogated by the electrodes.
Such methods and apparatus for detecting conductance changes would have many applications. For example, in planar gel electrophoresis, species such as proteins and DNA are separated in multiple tracks along with calibration species and are subsequently detected by staining. A method and apparatus for detecting conductance changes induced by species as they are separating would be desirable as they would not require staining, thereby saving resources, and could provide information about species as they separate in real-time. In chromatography, conductance detectors with large geometrical amplification factors could function as “universal” detectors as all species possess a dielectric constant. Also, they can provide superior sensitivity to charged species via conductivity measurements. Many such applications will be readily apparent to those skilled in the arts.
Therefore, it would be desirable to have a detector and method for detecting changes in conductance caused by a presence of a species in a sample mixture, such that the changes are detected with signal-to-noise that is enhanced by noise rejection means, are suitably insensitive to undesirable fluctuations caused by influences such as pressure, are amplified by suitably large geometrical amplification factors, and are compatible with fluidic systems having constrained geometries, such as capillaries and planar systems, such a planar electrophoresis. It is further desired that the detector be compatible with gradient elution and a variety of mobile phases, including water and mobile phases that may contain impurities, buffers, salts or other electrolytes.